Microcalorimetric Determination of Serum Triglycerides and Cholesterol

Abstract

Reaction heats measured in a microcalorimeter between varying amounts of human serum and 700 IU of lipase (EC 3.1.1.3) and those between serum and 8 U of cholesterol oxidase (EC 1.1.3.6) were significantly linear with the contents of triglycerides (r=1.00) and cholesterol (r=0.99), respectively. However, when sera from 13 human volunteers were subject to comparative study between the micro-calorimetric and spectrophotometric methods, the correlation became less appreciable (r=0.70 and r=0.71) presumably due to the individual variations in free glycerol and cholesterol ester-constituents that were deliberately omitted in the microcalorimetric method but were routinely included in the standard (multi-enzymatic) spectro-photometric clinical method. The contribution of interfering substances, if any, is not totally ruled out.     

Enzymatic Determination of Urinary Total 17β-Hydroxysteroids

Abstract

An enzymatic determination of urinary total 17β-hydroxysteroids is described. The principle of the method is as follows: after hydrolysis with β-glucuronidase and solvolysis, the ethyl acetate layer, which is washed with alkaline solution and water, is transferred to two test tubes, then each of them is evaporated. To one test tube is added 3β,17β-hydroxysteroid dehydrogenase (EC 1.1.1.51, from P. testosteroni), NAD⁺, 2-ρ-iodophenyl-3-ρ-nitrophenyl-5-phenyltetrazolium chloride (INT), and diaphorase (EC 1.6.99.2, from C. kluyveri) for determination of total 3β,17β-hydroxysteroids. To another test tube is added 3β-hydroxysteroid oxidase (EC 1.1.3.6, from B. sterolicum) for determination of total 3β-hydroxysteroids. The value of 17β-hydroxysteroids is calculated by subtracting the value for 3β-hydroxysteroids from the value for the 3β,17β-hydroxysteroids.     

Preparation and Properties of a Micro Enzyme Sensor for Creatine

Abstract

A micro enzyme sensor using creatine amidinohydrolase (EC 3. 5. 3. 3) and sarcosine oxidase (EC 1. 5. 3. 1) was developed for measuring creatine. The sensor consisted of platinum and silver wires fixed in a glass tubing with epoxy resin adhesive, and creatine amidinohydrolase and sarcosine oxidase immobilized on their tip by cross-linking method. The calibration curve for measurement of creatine, the effects of pH, temperature, the amount of flavin adenine dinucleotide and compounds on the sensitivity, and the life-time of the sensor were examined. Under optimal conditions, the response of the sensor was linear in the concontration range of 10 μ M ～ 0.65 mM creatine.     

The Successful Use of Cellulose Acetate Membrane for Very Low Density Lipoprotein Isolation and Cholesterol Quantitation

Abstract

The lipoproteins have been successfully isolated electrophoretically from the blood samples of patients having suffered from hyperlipoproteinemia type III but having survived myocardial infarction. The VLDL fraction from the cellulose acetate membrane is cut out and the total cholesterol and cholesterol of the VLDL extracted. The cholesterol is determined using an immobilized cholesterol esterase/oxidase column using a flow injection analysis system.     

Cholesterol oxidase biosensor based on a glassy carbon electrode modified with Nafion and methyl viologen

Cholesterol oxidase biosensor has been constructed by using bovine serum albumin and glutaraldehyde as cross linker to immobilize cholesterol oxidase and cholesterol esterase on a glassy carbon electrode modified with Nafion and methyl viologen. The biosensor has been used to determine total cholesterol in blood. The linear range of the determination is 2.5×10~7 to 1.0×10-4 mol/L. The detection limit is about 5.0×10~8 mol/L. The response time is 12 s. This biosensor has the advantage of high selectivity, sensitivity and short response time.     

Spectrofluorimetric assay for acyl CoA-cholesterol acyltransferase

A sensitive assay for acyl CoA-cholesterol acyltransferase (EC 2.3.1.26) in rat liver microsomal and mitochondrial preparations is described. The lowered cholesterol concentration in the enzyme reaction with oleoyl CoA is determined spectrofluorimetrically by using the cholesterol oxidase/peroxidase/p-hydroxyphenylpropionic acid system. The assay requires as little as 20 μg of protein in the enzyme preparation.     

Luminol Electrochemiluminescence-Based Biosensor for Total Cholesterol Determination in Natural Samples

ABSTRACT

A new cholesterol flow injection analysis biosensor is described. It is based on a luminol/hydrogen peroxide electrochemiluminescence (ECL) reaction induced by a glassy carbon electrode polarised at + 425 mV vs a Pt pseudo reference. The cholesterol b8543158 sensing layer is based on cholesterol oxidase (COD) immobilised on either UltraBind^TM membrane or Immunodyne membrane. The physicochemical properties of the UltraBind^TM type membranes enable a high-performance biosensor to be obtained. A high salt concentration, 3 M NaCl, is shown to induce a 400% increase of the immobilised COD activity, and intermediate salt concentrations, from 1 to 1.5 M NaCl, induce a 230% increase of the H₂O₂ sensor performances. Both phenomena enable the achievement of a sensitive ECL cholesterol biosensor which exhibited a 800% increase of its performance in the presence of 2 M NaCl. In those optimised conditions, the determination of free cholesterol could be performed with a detection limit of 0.6 nmol and a detection ranging over at least two decades.

When used in conjunction with a cholesterol esterase in solution, the biosensor enables the detection of free and total cholesterol in human sera, in 15 minutes with a good accuracy. The biosensor operational stability was satisfactory either with buffered standard solutions or with complex matrices since a total of 55 and 43 assays, respectively, could be performed with good reproducibility (CV = 4.8% and 8.3%) and without detectable loss of sensitivity.     

Direct Electrochemistry of Cholesterol Oxidase Immobilized on a Conducting Polymer: Application for a Cholesterol Biosensor

Direct electrochemistry of cholesterol oxidase (ChOx) immobilized on the conductive poly‐3′,4′‐diamine‐2,2′,5′,2″‐terthiophene (PDATT) was achieved and used to create a cholesterol biosensor. A well‐defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH₂ of ChOx, and the rate constant (k_s) was determined to be 0.75 s^?1. Glutathione (GSH) covalently bonded with PDATT was used as a matrix for conjugating AuNPs, ChOx, and MP, simultaneously. MP co‐immobilized with ChOx on the AuNPs‐GSH/PDATT exhibited an excellent amperometric response to cholesterol. The dynamic range was from 10 to 130 μM with a detection limit of 0.3±0.04 μM.     

Enzyme Electrode with Collagen-Immobilized Cholesterol Oxidase for the Microdetermination of Free Cholesterol

Abstract

Cholesterol oxidase has been immobililzed on collagen films associated to an electrochemical sensor to form a “cholesterol electrode”. The electrode poised at a potential of + 650 my vs Ag/AgCl detects the hydrogen peroxide produced in the enzymatic reaction. This device presents a very high sensitivity and a wide range of linearity (10^?7 M - 0.8.10^?4 M). The use of a non enzymatic electrode associated with the enzymatic one allowed the detection and correction of electrochemical interferences when applied to human sera for free cholesterol determination.     

Development and Application of an Immobilized Enzyme Electrode for the Determination of Sulfite in Foods and Feeds

Abstract

An enzyme electrode was developed using the enzyme sulfite oxidase (EC 1.8.3.1) immobilized onto pig intestine. All experimental parameters such as pH, temperature, buffer constituent concentration, etc., were thoroughly investigated and optimized when appropriate. Hydrogen peroxide was monitored amperometrically. The response to sulfite concentration was linear in the range of 5.2 × 10^?6 to 1.0×10^?3M. The proposed method was found to have a correlation coefficient of 0.952 when evaluated versus the standard titration method.     

Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system

Fabrication of an amperometric-rotating biosensor for the enzymatic determination of cholesterol is reported. The assay utilizes a combination of three enzymes: cholesterol esterase (ChE), cholesterol oxidase (ChOx) and peroxidase (HRP); which were co-immobilizing on a rotatory disk. The method is developed by the use of a glassy carbon electrode as detector versus Ag/AgCl/3 M NaCl in conjunction with a soluble-redox mediator 4-tert-butylcatechol (TBC). ChE converts esterified cholesterol to free cholesterol, which is then oxidized by ChOx with hydrogen peroxide as product. TBC is converted to 4-tert-butylbenzoquinone (TBB) by hydrogen peroxide, catalyzed by HRP, and the glassy carbon electrode responds to the TBB concentration. The system has integrated a micro packed-column with immobilized ascorbate oxidase (AAOx) that works as prereactor to eliminate l-ascorbic acid (AA) interference. This method could be used to determine total cholesterol concentration in the range 1.2 μM-1 mM (r = 0.999). A fast response time of 2 min has been observed with this amperometric-rotating biosensor. Lifetime is up to 25 days of use. The calculated detection limits was 11.9 nM. Reproducibility assays were made using repetitive standards solutions (n = 5) and the percentage standard error was less than 4%.     

Polypyrrole/multiwalled carbon nanotubes‐based biosensor for cholesterol estimation

The nanocomposite electrode comprising of polypyrrole (PPY) and carboxy functionalized multiwalled carbon nanotubes (MWCNT) has been electrochemically fabricated onto indium–tin–oxide (ITO) electrode using p‐toluene sulfonic acid (PTS). Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been immobilized onto this PPY– MWCNT/ITO nanocomposite electrode using N‐ethyl‐N‐(3‐dimethylaminopropyl) carbodiimide and N‐hydroxy succinimide chemistry for estimation of esterified cholesterol. The ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode has been characterized using Fourier transform infrared spectroscopy, electrochemical techniques, and scanning electron microscope. This ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode has a response time of about 9 s, linearity of 4 × 10^?4 to 6.5 × 10^?3 M/l of cholesterol oleate concentration, K_m of 0.02 mM, and thermal stability of upto 45°C. This electrode exhibits improved biosensing characteristics compared with other total cholesterol electrodes reported in literature till date and can be used to estimate cholesterol in blood serum samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Amperometric cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole films

Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8 V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 °C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 μA/mM and the apparent K_m value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3×10⁻³ S/cm.     

Derivatization of Ketosteroids for High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

A highly sensitive and selective derivatization of ketosteroids for use in high-performance liquid chromatography with electrochemical detection (HPLC/EC) is described. When the detector response for three phenylhydrazone derivatives was compared with one another using dehydroepiandrosterone, the p-nitrophenylhydrazone showed the highest sensitivity with a detection limit of 200 pg. Dehydroepiandrosterone and other principal 17-ketosteroids in human blood were quantitatively derivatized into p-nitrophenylhydrazones and efficiently separated by HPLC/EC on a μBondapak C_{1 8} column using 0.5%. NH₄H₂PO₄/methanol (2:7, v/v).     

Complex of tris(2-hydroxyethyl)amine with zinc bis[(2-methylphenoxy)acetate] as an aortic cholesterol esterase inhibitor in the experiment

The intramuscular administration of the complex of tris(2-hydroxyethyl)amine with zinc bis[(2-methylphenoxy)acetate] (Zitrimin), as an aqueous solution in a dose of 10 mg (kg of animal weight)^?1 for 2 months decreases activity of lysosomal lipolytic hydrolase (EC 3.1.1), cholesterol esterase (EC 3.1.1.13). Hence, the previously unknown feature of Zitrimin, that is, the ability to inhibit cholesterol esterase, attests to both structural and functional disorders of subcellular structures during development of atherosclerosis. The agents active towards such reactions can be useful in this case. Owing to the new properties, Zitrimin can be used to increase the vascular system stability to cholesterol during the atherosclerotic process. This expands the scope of applicability of this compound and gives prospects for development of new Zitrimin-based drugs for preventing the atherosclerotic vascular changes.

     

Construction of a N-Acetyl-L-methionine Electrode and Determination of Acylase with an Ammonia Gas Sensor

Abstract

The N-acetyl-L-methionine electrode is based on a coupled enzymatic system consisting of acylase and L-amino acid oxidase with an ammonia gas sensor; conditions of imobilization are optimized. N-acetyl-L-methionine in the range 4×10^?5–2×10^?3M gives a linear potential vs. log(concentration) plot with a response time of 2–5 min over the range specified. This electrode combined with an L-methionine electrode, based only on L-amino acid oxidase and an ammonia gas sensor, can be used for the determination of both substrates in mixtures, thus extending the feasibility of the method. Acylase (0.1–2.00) is determined in aqueous solutions by adding N-acetyl-L-methionine to the sample, and measuring the ammonia evolved with the gas-sensing electrode.     

HPLC Determination of Hepatic Cholesterol 7α-Hydroxylase Activity Using Immobilized Cholesterol Oxidase and Chemiluminescence Detection

Abstract

This paper describes an HPLC method for the determination of cholesterol 7α-hydroxylase activity, at high or low activity levels, that is sensitive and specific for 7α-hydroxycholesterol. The method relies on the generation of hydrogen peroxide by oxidation of 7α-hydroxycholesterol using the enzyme cholesterol oxidase which has been immobilized on porous glass beads. The hydrogen peroxide is subsequently detected by chemiluminescence generated by reaction of peroxide with bis-(2, 4, 6-trichlorophenyl)-oxalate (TCPO), a commonly used chemiluminescence reagent specific for peroxides. In the procedure, sample preparation is limited to extraction of the incubation mixture and injection of the concentrated extract.     

A Micro Planar Ampehometric Glucose Sensor Using an Isfet as a Reference Electrode

Abstract

A very small glucose sensor has been realized, which consists of a gold working electrode with a glucose oxidase immobilized membrane on it, and a gold counter electrode, all made on a sapphire substrate. By using the pH sensitive ISFET as a reference electrode, the potential for a solution, whose pH is constant, can be measured and irreversible metal electrodes, such as gold or platinum, can be used as working electrode and counter electrode. The sensor is very suitable for miniaturizing and mass production, because the Integrated Circuit (IC) fabrication process can be applied. The glucose oxidase immobilized membrane was also deposited by a lift off method, one of the IC processes. A glucose concentration, from 1 to 100 mg/dl, was measured with good linear current output.     

Metabolism Studies of Polycyclic Mutagens and Carcinogens Using Liquid Chromatography with Ultraviolet and Electrochemical Detection

Abstract

HPLC ultraviolet (UV) and electrochemical (EC) detectors connected in series were used to classify the metabolites of naphthalene, anthracene, acridine, and phenanthridine into phenolic and nonphenolic categories. This method of classification is based on comparison of the relatively high response of the EC detector to electroactive species such as phenols to the more nonselective response of the UV detector. The method is generally useful for many biologically active substances.     

Ferrocene and Ferricenium Ion as Versatile Photometric Titrants of H2O2 and D-Glucose in the Presence of Peroxidase and Glucose Oxidase. A Ferrocene-Peroxidase Stairway #

Abstract

Hydrogen peroxide can routinely be determined in the presence of ferrocene (FcH) and horseradish peroxidase by monitoring at 617 nm the enzymatically produced ferricenium dye. In contrast, D-glucose can be assayed by following the fading of the ferricenium dye FcH⁺PF₆ ^? in the presence of glucose oxidase. The change in absorbance in both cases corresponds to the amount of analyte. viz. H₂O₂ or D-glucose, in solution. The routine is very simple, invariant to the concentrations of both ferrocenes/ferricenium salt and enzyme and allows numerous “one pot” measuremeats with the detection limit of 10^?4 M for both the analytes. It takes 2–4 and 5–10 min to accomplish one analysis of H₂O₂ and D-glucose in the presence of peroxidase and glucose oxidase, respectively.